Freezing cells in blocking buffer? Urgent! - (Jan/17/2007 )

Hi all, I am performing immunofluoresce experiment. I fixed the cells in paraformaldehyde and pemeabilized the membrane with TritonX. Afterwards I continue with blocking the cells with PBS (2%BSA and 0.1%TritonX-100). My question is: What will happen if I keep the cells overnight in this blocking buffer at -20. Can I continue next day or sth would happen to the cells.I would be very glad for the answers.Thank you very muchCheers

-clementine-

I would leave the cells in blocking buffer in 4C. Some times for upto 10 days.

I have never left cells in blocking in -20C.

-scolix-

QUOTE (scolix @ Jan 17 2007, 08:42 PM)

I would leave the cells in blocking buffer in 4C. Some times for upto 10 days.

I have never left cells in blocking in -20C.

Do you think leaving them at -20 would burst the cells or so?

-clementine-

Crystals could form and might damage the cells such that the cellular morphology could b affected.

But honestly, I am not sure. IF u have the cells in -20C, just go ahead and complete it and observe how the cells looks like. might tell us something.

-scolix-

In my experience cell freezing leads to very high background fluorescent signal, I don't recomend you do it under PFA-fixation conditions. If you want to save your sample you can leave it at RT or 4 0C overnight and even longer without any troubles. Usually I stain cells overnight at 4 0C and have very law background

-Koshechan-

So here comes the result of the experiment. The cells frozen in blocking buffer can be nicely processed afterwards and analyzed by immunofluorescence.But one thing I did was to rapidly thawing them at 37C incubator so that no crystals form.Thanks to all for the ideas.Cheers

-clementine-

Good to know it worked.

enjoy !!!

-scolix-

QUOTE (clementine @ Jan 17 2007, 07:29 PM)

Hi all, I am performing immunofluoresce experiment. I fixed the cells in paraformaldehyde and pemeabilized the membrane with TritonX. Afterwards I continue with blocking the cells with PBS (2%BSA and 0.1%TritonX-100). My question is: What will happen if I keep the cells overnight in this blocking buffer at -20. Can I continue next day or sth would happen to the cells.I would be very glad for the answers.Thank you very muchCheers

I have never tried but I would fear that part of BSA will not solve again and remain precipitated which may result in artefacts after staining